Synapses are highly specialized cell-cell junctions that mediate communication between neurons. These structures are composed of pre- and post-synaptic terminals and are the basis for the complex circuitry found in the brain. Most postsynaptic terminals of excitatory synapses take the form of dendritic spines, which are actin-rich protrusions that emanate from the dendrite shaft. Not surprisingly, the formation and plasticity of dendritic spines and synapses play a central role in cognitive function and abnormalities in these structures are associated with a number of neurological disorders. Despite the importance of spines and synapses in the central nervous system, the molecular mechanisms that regulate the formation of these structures are not well understood. A limitation toward identifying key molecules that regulate spine and synapse formation has been the great difficulty in observing synapses as they form. We are developing novel microfluidic devices that will allow us to dynamically observe forming synapses (Specific Aim I). Several innovations in the design of these devices will significantly enhance our ability to image the early steps of synapse formation with high spatial and temporal resolution. In Specific Aim II, we will apply this technology to examining the spatiotemporal dynamics of actin during synaptic assembly. In addition, we will test our hypothesis that the activity of Rho family GTPases, which are key regulators of actin, is critical in the initial assembly and maturation of synapses. For these experiments, we will use cutting-edge microscopy technologies, including FRAP, photoactivation, and FRET to examine actin dynamics and regulation during synapse formation. The development of these microfluidic platforms will be of great interest and benefit to neurobiologist by providing a platform for identifying the key molecular signals that regulate the assembly of synapses.